Responding to and repairing DMA damage is critical for cell viability and disease prevention of the organisms. Cells have evolved complex DMA damage response mechanism to counteract with various forms of external and internal DMA damaging agents that are hazardous to the genome. Many key components of the DNA damage response quickly localize to damage sites following exposure of cells to genotoxic stress, responsible for DNA damage signaling and repair. In this proposal, we are aiming to identify additional such proteins by taking a genomic approach to identify proteins that form DNA damage induced foci. We propose to use retroviral GFP protein-trapping vectors to tag endogenous proteins with GFP and genome-widely identify GFP fusion proteins that form DNA damage induced foci. This will greatly facilitate our understanding of the DNA damage signaling and repair pathway. In addition, we are proposing to identify proteins that are recruited to the DNA damage sites by purifying proteins that associate with the phosphorylated H2AX. H2AX plays a critical role in DNA damage response, responsible for proper assembly of many DNA damage signaling/repair proteins. We are also attempting to address the regulation and biological significance of foci formation of the DNA damage mediators/adaptors 53BP1 and MDC1, using biochemical and genetic approaches. Together, by accomplishing the specific aims of this proposal, we will gain a clearer picture of how cells respond and repair DNA damage.